脂質代謝會參與壽命的調節，從實驗室先前的研究得知視網膜退化A基因 (rdgA; 二酰甘油激酶) 及視網膜退化B基因 (rdgB; 磷脂酰肌醇轉移蛋白) 兩者均可在果蠅中透過減少雷帕黴素標靶蛋白質訊號傳遞路徑 (TOR signaling pathway) 而延長壽命並增加其對氧化壓力的耐受度。在磷酸肌醇轉換循環 (phosphatidylinositol turnover) 中，磷脂酰肌醇合成酶 (Pis) 位於磷脂酰肌醇轉移蛋白上游，功能為將胞磷酸二磷酸二酰甘油 (cytidine diphosphate diacylglycerol) 生成磷脂酰肌醇 (phosphatidylinositol, PI)，使轉移蛋白能有充足的PI運送出內質網進行磷酸化作用。目前對於Pis是否能調節壽命仍未知曉。本篇論文中，我們發現Pis表現量較低的果蠅突變株 PisB33580 的壽命有延長現象，同時對於氧化壓力耐受度也有增強的情形。在神經中降低Pis表現量也能延長壽命，然而過度表現Pis並不會影響果蠅壽命。藉由西方墨點法我們觀察到不管在Pis突變株還是透過RNAi特定降低神經中Pis表現量的果蠅中，磷酸化S6激酶 (p-S6K) 及磷酸化蛋白激酶B (p-AKT) 的量皆會下降，另外在利用RNAi降低Pis表現量的果蠅中還發現LC3II表現量上升的情形。這些結果顯示雷帕黴素標靶蛋白質及胰島素/ IGF-1訊號傳遞路徑皆可能參與由低Pis表現量所調控的壽命現象。在Pis基因組區域上游及第一個內含子中找到三個轉錄因子dFOXO的潛在結合位，初步認為dFOXO可能透過減弱胰島素/ IGF-1訊號傳遞路徑而參與Pis調控的壽命現象。總合來說，這些數據顯示了減少Pis表現量可能會透過雷帕黴素標靶蛋白質及胰島素/ IGF-1兩種訊號傳遞路徑來調節果蠅的壽命。

Lipid metabolism plays a role in lifespan regulation. Our previous studies reported that knockdown of rdgA (retinal degeneration A, as known as diacylglycerol (DAG) kinase) and rdgB (retinal degeneration B, phosphatidylinositol transfer protein) both can extend lifespan and enhance oxidative stress tolerance via reduced TOR (target of rapamycin) signaling in Drosophila. Phosphatidylinositol synthase (Pis) is upstream of rdgB and produces phosphatidylinositol from cytidine diphosphate diacylglycerol in phosphoinositide turnover cycle. Whether Pis can regulate lifespan remains unknown. Here, we report that a mutant with lower Pis expression exhibits extended lifespan and enhanced resistance to oxidative stress. Tissue-specific knockdown of Pis in neurons also reproduces longevity, however overexpression of Pis does not affect lifespan. By western blotting, we observe reduced levels of phosphorylated-S6K and phosphorylated-AKT in the Pis mutant and RNAi knockdown longevity flies, as well as we also found increased levels of LC3II in the Pis RNAi knockdown flies. Both suggest TOR and insulin/IGF-1 signaling pathways may participate in the Pis-knockdown-mediated longevity. Interestingly, we find that there are three transcription factor potential binding sites in the upstream and the first intron of Pis genomic region, suggesting that dFOXO may play a role in Pis-mediated lifespan regulation via reduced insulin/IGF-1 signaling. In summary, our results uncover a novel function of Pis in regulating longevity possibly via TOR and insulin/IGF-1 signaling pathways in Drosophila.

Abstract i
中文摘要 ii
致謝 iii
Table of Content 1
Introduction 2
Materials and methods 6
Fly strains and maintenance 6
Lifespan survival measurement 6
Oxidative resistance test 7
RNA extraction and Reverse transcription 7
Quantitative Real-time PCR (Q-PCR) 8
Immunoblot assay 8
Results 10
The mechanism of rdgA- and rdgB-mediated longevity are in the same way 10
A mutant with decreased phosphatidylinositol synthase expression exhibits lifespan extension 10
Specific knockdown of Pis in neurons promotes longevity in Drosophila 11
Better oxidative stress resistance was detected in the Pis mutant but not in the Pis RNAi knockdown lines 11
Overexpression of Pis in neurons do not alter lifespan in Drosophila 12
Lower p-S6K levels and higher LC3II levels are detected in Pis reduced flies 13
Reduced p-AKT levels are detected in the Pis mutant and RNAi lines 13
dFOXO may participate in Pis-knockdown-mediated longevity 14
Discussion 15
Figures 18
Tables 40
References 49

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